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A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

Gornicka A, Bragoszewski P, Chroscicki P, Wenz LS, Schulz C, Rehling P, Chacinska A - Mol. Biol. Cell (2014)

Bottom Line: We identified a transient interaction between our model substrates and Tom40.Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22.Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

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Effect of TOM channel modification on the import of mitochondrial precursor proteins. (A) Steady-state protein levels of mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were modified with mPEG and analyzed by reducing SDS–PAGE, followed by immunodecoration. (B) Native migration of the TOM complex upon modification with mPEG of the mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were analyzed by BN-PAGE and immunodecoration with anti-Tom40 antibody. (C) Radiolabeled F1β was imported into mitochondria isolated from cells that carried Tom40, Tom40C89/C360, or Tom40C130/C138 upon modification with mPEG. (D) Radiolabeled Cox19 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (E) Radiolabeled Tim13 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (F) Radiolabeled Mix17 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (C–F) The samples were treated with proteinase K and analyzed by nonreducing or reducing SDS–PAGE. WT, wild-type; IA, iodoacetamide; Δψ, electrochemical potential. (D–F) Quantitations of 35S-radiolabeled precursor import (bottom). Import into WT mitochondria after 40 (D), 27 (E), or 15 min (F) was set to 100%. SEM of three independent experiments.
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Figure 3: Effect of TOM channel modification on the import of mitochondrial precursor proteins. (A) Steady-state protein levels of mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were modified with mPEG and analyzed by reducing SDS–PAGE, followed by immunodecoration. (B) Native migration of the TOM complex upon modification with mPEG of the mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were analyzed by BN-PAGE and immunodecoration with anti-Tom40 antibody. (C) Radiolabeled F1β was imported into mitochondria isolated from cells that carried Tom40, Tom40C89/C360, or Tom40C130/C138 upon modification with mPEG. (D) Radiolabeled Cox19 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (E) Radiolabeled Tim13 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (F) Radiolabeled Mix17 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (C–F) The samples were treated with proteinase K and analyzed by nonreducing or reducing SDS–PAGE. WT, wild-type; IA, iodoacetamide; Δψ, electrochemical potential. (D–F) Quantitations of 35S-radiolabeled precursor import (bottom). Import into WT mitochondria after 40 (D), 27 (E), or 15 min (F) was set to 100%. SEM of three independent experiments.

Mentions: We investigated whether TOM is engaged in a possible alternative route for MIA-dependent proteins. The core of the TOM complex is formed by the β-barrel protein Tom40, which forms a channel for protein import (Pfanner et al., 2004; Neupert and Herrmann, 2007; Schmidt et al., 2010; Endo et al., 2011; Dimmer and Rapaport, 2012; Qiu et al., 2013). Cysteine residues that are positioned such that they face the lumen of the channel should be amenable to chemical modifications, which would create spatial hindrance and clog the Tom40 channel. On the basis of a recent study (Qiu et al., 2013), we used two yeast strains that harbor Tom40 with the cysteine residues introduced in positions 89/360 and 130/138, in addition to the wild-type strain and a strain with Tom40 that lacked native cysteine residues (Tom40CFREE; Supplemental Figure S1A). The levels of mitochondrial proteins in the mutant strains were unaffected (Supplemental Figure S1B). We blocked the Tom40 channel by applying the alkylating agent methoxypolyethylene glycol maleimide (mPEG; molecular weight, 5 kDa). This compound reacts with accessible cysteine thiol groups. In intact mitochondria, mPEG modified cysteine residues of the Tom40 mutants (Figure 3A, lanes 4 and 8) but did not affect Tom40CFREE or wild-type Tom40 with native cysteine residues, indicating that native cysteine residues were not accessible for modification (Figure 3A, lanes 2 and 6). Given the presence of cysteine residues in the cytosolic domain, Tom70 was shifted due to mPEG modification, whereas Tom22 and Tom20 remained unmodified (Figure 3A). The mPEG modification of the Tom40 mutants with the thiol groups facing the channel resulted in a change in TOM complex migration in blue-native gel (Figure 3B, lanes 4 and 8). These experiments verified the specificity of mPEG treatment.


A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

Gornicka A, Bragoszewski P, Chroscicki P, Wenz LS, Schulz C, Rehling P, Chacinska A - Mol. Biol. Cell (2014)

Effect of TOM channel modification on the import of mitochondrial precursor proteins. (A) Steady-state protein levels of mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were modified with mPEG and analyzed by reducing SDS–PAGE, followed by immunodecoration. (B) Native migration of the TOM complex upon modification with mPEG of the mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were analyzed by BN-PAGE and immunodecoration with anti-Tom40 antibody. (C) Radiolabeled F1β was imported into mitochondria isolated from cells that carried Tom40, Tom40C89/C360, or Tom40C130/C138 upon modification with mPEG. (D) Radiolabeled Cox19 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (E) Radiolabeled Tim13 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (F) Radiolabeled Mix17 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (C–F) The samples were treated with proteinase K and analyzed by nonreducing or reducing SDS–PAGE. WT, wild-type; IA, iodoacetamide; Δψ, electrochemical potential. (D–F) Quantitations of 35S-radiolabeled precursor import (bottom). Import into WT mitochondria after 40 (D), 27 (E), or 15 min (F) was set to 100%. SEM of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: Effect of TOM channel modification on the import of mitochondrial precursor proteins. (A) Steady-state protein levels of mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were modified with mPEG and analyzed by reducing SDS–PAGE, followed by immunodecoration. (B) Native migration of the TOM complex upon modification with mPEG of the mitochondria isolated from cells that carried Tom40, Tom40CFREE, Tom40C89/C360, or Tom40C130/C138. The samples were analyzed by BN-PAGE and immunodecoration with anti-Tom40 antibody. (C) Radiolabeled F1β was imported into mitochondria isolated from cells that carried Tom40, Tom40C89/C360, or Tom40C130/C138 upon modification with mPEG. (D) Radiolabeled Cox19 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (E) Radiolabeled Tim13 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (F) Radiolabeled Mix17 was imported into mitochondria isolated from cells that carried Tom40 or Tom40C130/C138 upon modification with mPEG. (C–F) The samples were treated with proteinase K and analyzed by nonreducing or reducing SDS–PAGE. WT, wild-type; IA, iodoacetamide; Δψ, electrochemical potential. (D–F) Quantitations of 35S-radiolabeled precursor import (bottom). Import into WT mitochondria after 40 (D), 27 (E), or 15 min (F) was set to 100%. SEM of three independent experiments.
Mentions: We investigated whether TOM is engaged in a possible alternative route for MIA-dependent proteins. The core of the TOM complex is formed by the β-barrel protein Tom40, which forms a channel for protein import (Pfanner et al., 2004; Neupert and Herrmann, 2007; Schmidt et al., 2010; Endo et al., 2011; Dimmer and Rapaport, 2012; Qiu et al., 2013). Cysteine residues that are positioned such that they face the lumen of the channel should be amenable to chemical modifications, which would create spatial hindrance and clog the Tom40 channel. On the basis of a recent study (Qiu et al., 2013), we used two yeast strains that harbor Tom40 with the cysteine residues introduced in positions 89/360 and 130/138, in addition to the wild-type strain and a strain with Tom40 that lacked native cysteine residues (Tom40CFREE; Supplemental Figure S1A). The levels of mitochondrial proteins in the mutant strains were unaffected (Supplemental Figure S1B). We blocked the Tom40 channel by applying the alkylating agent methoxypolyethylene glycol maleimide (mPEG; molecular weight, 5 kDa). This compound reacts with accessible cysteine thiol groups. In intact mitochondria, mPEG modified cysteine residues of the Tom40 mutants (Figure 3A, lanes 4 and 8) but did not affect Tom40CFREE or wild-type Tom40 with native cysteine residues, indicating that native cysteine residues were not accessible for modification (Figure 3A, lanes 2 and 6). Given the presence of cysteine residues in the cytosolic domain, Tom70 was shifted due to mPEG modification, whereas Tom22 and Tom20 remained unmodified (Figure 3A). The mPEG modification of the Tom40 mutants with the thiol groups facing the channel resulted in a change in TOM complex migration in blue-native gel (Figure 3B, lanes 4 and 8). These experiments verified the specificity of mPEG treatment.

Bottom Line: We identified a transient interaction between our model substrates and Tom40.Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22.Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

Show MeSH